EFFICACY OF ESSENTIAL OILS ON GERMINATION PERCENTAGE AND GROWTH OF DURUM WHEAT IN COMPARISON OF PESTICIDE: TEBUCONAZOLE
https://doi-004.org/6812/17633617778284
Bouchiha Hanene 1, Rizi Aicha 2, Acidi Anisa2, Attia Moussa3 , Mecheri Hind1.
1 Department of Applied Biology, Echahid Cheikh Larbi Tebessi University, Tebessa, Algeria.
2Department of chemistry, Badji Mokhtar University, Annaba, Algeria.
3 Instituteof Mines, Echahid Cheikh Larbi Tebessi University, Tebessa, Algeria.
hanene.bouchiha@univ-tebessa.dz
Submission date: 01.01.2025. Accepted 01.05.2025 Publicaion.12.06.2025
Abstract:
Cereals and their derivatives are the backbone of the world’s diet. Durum wheat, in particular, is one of the most important food sources in most countries, which is why studies have always been developed to improve the quality and quantity of its production.
In this regard, we have undertaken work to reduce the harmful effects of the fungicide (Tebuconazole) used by the Algerian Inter-professional Cereals Office (OAIC) to treat wheat seeds in the form of a concentrated suspension containing 60 g/l of Tebuconazole (with approved use of 50 ml/quintal diluted in 550 ml of water) by essential oils (HES) extracted from Mentha piperita as a natural antioxidant provided with different doses (0.05µL/mL, 0.1µL/mL and 0.15µL/mL)on the growth, germination of seeds and some wheat metabolites in Petri dishes of 09 cm diameter throughout 14 with 03 repetitions.
The results obtained indicate that the HES extract of Mentha piperita has a positive effect on the growth of roots and stems of durum wheat and on protein synthesis in comparison with the fungicide Tebuconazole.
Therefore, it is worth noting that mint HES extract has a strong antioxidant effect on the adverse impact of Tebuconazole on durum wheat germination.
Keywords: Cereal, Fungicide, Tebuconazole, Essential oils, Mentha piperita.
Introduction :
Cereal production, particularly wheat, is a primary global concern. Demand in the Mediterranean for 2020 is estimated at over 111 million quintals (Hervieu and al., 2006). Residents of Arab countries appear to be the largest consumers worldwide. Nearly 700 grams per person per day in Morocco, approximately 600 grams in Tunisia, Algeria, and Egypt, compared to 400 grams in India and 320 grams in France (Abis, 2012).
Cereals have always been and remain an essential commodity in the diet. Cereal farming is one of the main activities in Algeria, particularly in arid and semi-arid areas (Ducellier, 1931). It is estimated that approximately 3.5 million hectares of agricultural land are devoted to cereal cultivation; this was confirmed by the 2017 statistics of the ONFAA (National Observatory of Agricultural and Agronomic Sectors), 40% of which is occupied by durum wheat (Benabdelkader and Nouar, 2018).
Most wheat is used primarily for human consumption, thanks to its high protein content and the presence of gluten, which gives pasta better cooking properties. Moreover, the production of pasta, leavened and unleavened bread, couscous, and other traditional foods is commonly associated with durum wheat (Graziano and al., 2019). Straw is also used as animal feed (Debiton, 2010).
In recent years, this cereal has also been used in the industrial sector to produce biofuel and bioplastics based on gluten or starch. The main uses are plastic bags, agricultural plastics, packaging, and certain hygiene products.
In the pharmaceutical industry, processed starch could be used as a sugar-coating agent, binder, or even an active ingredient such as sorbitol. In smaller proportions, processed starch can also be used in the manufacture of paper, cardboard, and detergents (Debiton, 2010).
The importance of cereals’ economic and nutritional role means that every country in the world must protect itself and ensure a minimum of food security. (Bourihane and Mekkaoui, 2013). To meet the growing demands of the population, farmers resort to intensifying cereal crops. However, these practices are accompanied by the appearance of several diseases caused by pathogenic fungi. These attacks can lead to significant quantitative and qualitative damage to wheat (Meksem and al., 2007; Hennouni and al., 2008).
To prevent these fungal diseases,cereal growers regularly and systematically apply systemic fungicides to seeds before cultivation. Their use thus meets agronomic and economic imperatives (Schreck, 2008). However, the continuous and repeated use of identical families of fungicides or pollutants can have unintended and unwanted effects on plants (Dhanamanjuri and al., 2013). The damage caused to plants by these treatments can manifest through visible marks on germination, including a slowdown in growth. They can also disrupt biochemical activity, mainly synthesizing carbohydrates and amino acids (Borjiba and Ketif, 2009).
According to Bouziani (2007), Algeria is one of the countries that uses the most significant quantities of pesticides. Tebuconazole is among the most widely used fungicides (approximately 25% of the fungicide market) (Leroux, 2003; Leroux, 2005) in more than a hundred countries, including Algeria, as a seed treatment (Asrar and al., 2004).
In this regard, we have undertaken this work, whose aim is to use HES extracted from Mentha piperita as a natural antioxidant to reduce the harmful effects of Tebuconazole on germination, growth, and biochemical parameters such as total protein levels of durum wheat Triticum durum (in vitro after 7 and 14 days of treatment).
Durum wheat :
Wheat, barley, rye, and oats constitute the first major cereals category. Wheat is an annual, herbaceous, monocotyledonous plant belonging to the genus Triticum of the grass family (Anne-Laure, 2007). Among these cereals, durum wheat (Triticum durum) is one of the oldest species and constitutes a large part of the diet.
Table 01 : The systematic position of wheat (Chadefeau and Emberger, 1960):
| Branch | Phanerogams |
| Sub-branch | Angiosperms |
| Phylum | Liliiflores |
| Order | Germinates |
| Family | Graminaceae |
| Subfamily | Festucoids |
| Gender | Triticum |
Today, two species dominate production:
– Soft wheat (Triticum aestivum): which represents more than 90% of world production
– Durum wheat (Triticum durum) constitutes 5% of it and is traditionally cultivated in the Mediterranean basin (Gooding, 2009).
The Middle East, the Near East, and North Africa are considered the centers of origin and diversification of durum wheat (Vavilov, 1951). In Algeria, the area devoted to durum wheat can be estimated at 1,200,000 hectares. It is an indigenous crop par excellence because it is particularly adapted to the environment, heat, and lack of humidity (Jean Blottière, 1930).
Tebuconazole :
Tebuconazole is a chemical compound belonging to the triazole family. Relatively lipophilic, it is poorly soluble in water (36 mg/L). Having a relatively moderate risk of volatilization (KH = 1×10-5 Pa.m3/mol at 25°C), this compound, solid at room temperature, is very stable to hydrolysis and photolysis. (Cosic and al., 2006).
Table 05: Chemical composition of Tebuconazole (Couvreur, 2002)
| Fungicide | Chemical Family | Chemical Structure |
| Tebuconazole | Triazole | 1-(4-chlorophenyl)-4,4-dimethyl-3-(1H-1,2,4-triazole-1-ylmethyl)pentane-3-ol) |
Tebuconazole is a widely used fungicide worldwide. It is an active ingredient in plant protection and biocidal products, and it is used for its antifungal properties.
Tebuconazole treats wheat seeds in Algeria at the Algerian Interprofessional Office of Cereal Growers (OAIC). It is intended for various cereal smuts, with a protective, curative, and eradicating action, as well as various pathogens such as Fusarium sp, Puccinia sp, Rhynchosporium sp, Pyrenophora sp, and Septoria sp (Rivale, 2010). The efficacy of Tebuconazole against wheat bunts has been demonstrated by Rajkovič and Dolovac (2006) and Nagy and Moldovan (2006 and 2007).
Seed treatment with the latter ensures the protection of seed surfaces (the low solubility of the active ingredient in water makes it available near the seed for slow root absorption). These substances penetrate during the imbibition phase through the seed coats for protection against pathogens that affect the interior of the seeds. Then, they are transported to the young stem by translocation through the vascular tissues and are effective until the three-leaf stage (Rajkovič, Dolovac, 2006).
Phytotoxicity of Tebuconazole :
Phytotoxicity is the impact or damage a compound can cause on specific plant characteristics (germination, growth, and development of roots and stems) (Moore and Kröger, 2010). Phytotoxicity problems related to systemic treatments, particularly on seeds, have been widely
Studied, highlighting the inhibition of seedling emergence and development. Venancio and al. (2003) suggested that metabolic changes may occur during fungicide absorption unrelated to defense against fungi. These effects are all the more accentuated when these fungicides are applied excessively (Hallam, 2010). This is the case for wheat; Siddiqui and al. (2001) reported the inhibitory effect of the fungicide methyl thiophanate on germination, growth, and accumulation of root and stem biomass. As tebuconazole is relatively persistent in different compartments of the environment, it can constitute an ecological risk despite its low mobility in soils. Tebuconazole is, however, very often detected in the influents and effluents of many European wastewater treatment plants. Its biodegradability is very low, even though some bacterial and fungal strains can biotransform this fungicide efficiently.
Many authors have also been interested in the impact of tebuconazole on soil microbial communities. The results obtained are pretty contradictory: Strickland and al. (2004) and Bending and al. (2007) observed no effect on microbial biomass after addition of tebuconazole at a concentration of 1 and 5 mg.kg-1 respectively, while Cycon and al. (2006) only found a reduction in this biomass after addition of a high concentration (270 mg.kg-1) and Munoz-Leoz and al. (2011) noted a systematic reduction, from the addition of 5 mg.kg-1 of tebuconazole, with a decrease that was all the more significant as the tebuconazole concentration was high.
Materials and methods :
Our work was conducted at the University of Tébessa (Algeria) in the applied biology laboratories.
1. Study materials :
1.1 Plant material studied :
- Durum wheat (Triticum durum) :
The material used in our work is an edible wheat Triticum durum plant in the GTA variety.
*Origin :
The durum wheat variety GTA durum was produced in Cimmy (Mexico) and comes from the Interprofessional Cereals Office (OAIC) and specifically from the CCLS (Cooperative of Cereals and Dried Vegetables) processed by the fungicide (Tebuconazole) in the form of a concentrated suspension containing 60 g/l of Tebuconazole with approved use of 50 ml/quintal diluted in 550 ml of water.
1.1.2 Peppermint (Mentha piperita) :
* Extraction of essential oils from mint :
HES were obtained by hydrodistillation of the aerial part of Mentha piperita. 100 g of mint leaves were cut into small pieces and placed in a 1000 ml flask with 600 ml water. (Bouchiha H., 2016)
* Conservation of the essential oils obtained :
The conservation of HES requires certain essential precautions. This is why we have stored them at around 4°C in a hermetically sealed brown glass bottle to protect them from air and light oxidation. (Bouchiha H., 2016)
1.2 Chemical material studied :
Tebuconazole is a fungicide designed to treat cereal seeds. It has a systemic action on many fungi and effectively combats cereal seed diseases. – Wheat bunt (Tilletia caries) – Loose smut of wheat (Ustilago nuda).
2. Cultivation and processing of seeds :
Wheat seeds were grown according to the method described by Kaur and Duffus (1989). Ten seeds were randomly selected and placed in 9 cm diameter Petri dishes on blotting paper, soaked with 8 ml of water.
The seed treatment was carried out using a solution prepared from a mixture of HES of Mentha piperita and water over 14 days according to the doses: (0 ul/ml, 0.25 ul/ml, 0.5 ul/ml, and 1 ul/ml) with 3 repetitions for each Petri dish (watering every 2 days with 8 ml of the prepared solutions).
3. Measurement of germinative parameters :
3.1 Average germination rate :
It is expressed as the percentage of germinated seeds in relation to the total number of seeds per petri dish. Seed germination is considered positive when the rootlets reach 5 mm in length (Kaur and Duffus, 1989).
3.2 Average root length :
The seeds are carefully removed from the Petri dishes using a pencil. The ends of each root are marked, and their length is then measured after 7 and 14 days of the experiment.
3.3 Average length of stems :
The length of the stems was measured using a ruler after 7 and 14 days of the experiment.
4. Biochemical dosages :
4.1 Total protein dosage :
Protein dosage is carried out according to the Bradford method (1976), which is based on the color change of the BBC pigment (Coomassie Brilliant Blue) from red to blue in the presence of the protein.
– Weigh 100mg of fresh green leaves from each sample.
– Grinding fresh green leaves with 5 ml of distilled water in a mortar
– filter the macerate.
– Take 0.2ml of solution, then add 0.2ml of BBC reaction and 1.6ml of distilled water.
– Vortex the solutions and leave the solutions for 5 minutes to 1 hour.
– Optical density reading at wavelength 595 min.
The total protein content is determined by referring to the calibration curve drawn from known BSA concentrations.
Results:
Effects of different doses of Mentha piperita HES on germination and growth :
Effects on percentages of germination :
Germination is a physiological process defined as the sum of events that lead the dry seed to germinate; it begins with the intake of water and ends with the elongation of the embryonic axis.
That is to say, it is the passage from the seed’s latent life to its active life under the effect of favorable factors. According to Heller and al. (2004), a seed has germinated when the radicle pierces the envelopes or is visibly elongated.
Figure 2: Effects of Mentha piperita essential oils on the germination percentage of wheat seeds compared with ACIL-treated seeds.
Effects HES on average root length :
The variations in the average root lengths of wheat seeds by different doses of mint HES are represented in the following figure:
Figure 3: Effects of Mentha piperita HES on mean wheat root length after 7 days of treatment compared with ACIL-treated seeds.
The average length of the roots of wheat seeds treated with ACIL is 3.6 cm. This value is low compared to those after treatment with essential oils: 5.3 with the dose (0.15µL/mL), 4.6 with the dose (0.1µL/mL), and finally 3.8 with the dose (0.05µL/mL).
Figure 4: Effects of Mentha piperita essential oils on average wheat root length after 14 days of treatment compared with ACIL-treated seeds.
From the figure above, we can see that the seeds treated with ACIL developed roots with an average length of 6.3 cm. Thus, contact with mint HES increases this average: for the dose0.1µL/mL, the value was recorded(7.4 cm), while for the dose0.15µL/mL, the value was marked(8.7 cm).
Effects HES of mint on the average length of the stems :
Figure 5: Effects EO of Mentha piperitaon on the average length of wheat stems after 7 days of treatment compared with seeds treated with ACIL.
Figure 6 : Effects of Mentha piperita EO on the average length of wheat stems after 14 days of treatment with seeds treated with ACIL.
It is observed that the control seeds developed stems with an average length of 5.4 cm, and contact with mint EO causes an increase in this average with an increasing dose for the different doses. (0.15µL/mL), (0.1µL/mL) and the dose (0.05µL/mL).
Effects of EO on biochemical parameters of germination :
Effects on stem protein content :
Figure 7: Effects of different doses of Mentha piperita EO on total protein content (ug/g of MF).
The figure above represents the effects of different doses of Mentha piperita on the total protein content (ug/g of MF). A significant increase was recorded, especially with the doses (0.15µL/mL) and (0.1µL/mL).
Discussion :
The fungicidal activity of triazoles is based on their ability to inhibit the biosynthesis of ergosterol, an essential membrane sterol of fungal strains, by binding to the heme of cytochrome P450 (CYP51 = lanosterol 14α-dimethylase) involved in the transformation of lanosterol into ergosterol (Roberts and Hutson, 1999; Onyewu and al., 2003).
This inhibition leads to an accumulation of non-demethylated sterols, modifying the shape and physical properties of the fungal membrane (decreased fluidity) and leading, in particular, to a change in permeability and poor functioning of membrane proteins. The main objective of this experiment is to know the effect of EO compared to the fungicide studied on the growth, morphology, and composition of durum wheat. We learned some growth parameters expressed by the germination rate, the average length of root stems, and the protein dosage.
The results obtained show that the EO of mint used with the dose0.15µl/increased the germination percentage (90%) compared to the dose0.1µL/ml (60%)and dose0.05µL/ml (50%)and in comparison with the control (40%).
These results are consistent with those obtained by Siddiqui and Zaman (2004) following the application of high concentrations of benomyl on maize. Similar results were observed in rice after the application of fungicides of the benzimidazole and thiophanate family (Ibiam and al., 2008) and in sorghum after treatment with carbendazim (Avinash and Hosmain, 2012).
Several species (Glycine max, Allium cepa, Pennisetum americanum, Brassica compestris, Vigna radiata, Capsicum annum, Theobroma cacao) have revealed their sensitivity to these treatments through a reduction in germination and growth in the face of increasing concentrations of fungicides applied (Aksoy and Deveci, 2012). According to several authors, reduction of germination and growth is considered an adaptation strategy to stress (Prado and al., 2000). Zhu (2001) suggests that reduced growth is a good indicator of the toxicity of seedling tissues, which may be due to the conservation of energy for use in the defense response and/or to reduce the risk of damage.
Ahmad and al., 2012 go against these results; they attributed the reduction of growth to substituting pollutants with metabolic enzymes. The hypothesis is supported by many authors who have shown the effect of pesticides on several hydrolytic enzymes responsible for starch degradation during germination (Chibi and Sayah, 2011).
Our results are consistent with those of many authors who have reported negative effects of high concentrations of seed fungicides on the germination of wheat (Siddiqui and al., 2001), maize (Siddiqui and Zaman, 2004), and rice and sorghum (Avinash and Hosmain, 2012). The results of the germination percentage lead us to believe that the toxicity of fungicides is immediately present, even before the appearance of the first roots, during the germination process (Meksem, 2007).
Contrary to the results of Taye and al. (2013), which showed that seed treatments with certain fungicides could accelerate their germination. Rangwala and al. (2013) also reported that the fungicide Carbendazim at high concentrations would improve wheat germination.
Our growth and vigor results showed that fungicide treatments reported inhibitory effects compared to treated ones. This aligns with the results of Smiley and al. (1996) and Dhanamanjuri and al. (2013). Our results align with the authors cited previously, who indicate that roots seem more affected than stems by the application of fungicides.
Meksem (2007) obtained that there were more significant reductions in root growth compared to stems after the application of the fungicides propiconazole and fluquinconozole in wheat. According to this same author, the reduction in root length observed in our experiments is explained by the inhibition of their development caused by the absorption of the fungicide’s active ingredient by the wheat grains.
According to Prasad (1995), reduced root elongation is often indicative of toxicity. Zhu (2001) suggests that growth problems may be due either to conservation of energy for use in defense response and/or to reducing the risk of damage. Gopi and al. (2005) explained the reduction in growth by inhibiting auxin production, reflecting disruption of division and elongation of meristems.
(Epron and al., 1999)showed that chemical treatments cause moderate to high salinity, which can strongly impair root elongation by salinity, which may be due to the inhibition of cell extension following the decrease in turgor. The same results were observed at the level of stem growth (William and Hapkin.,1999).
General conclusion :
The results obtained during this study concerning the influence of Tebuconazole used by the OAIC show its negative impact on wheat germination and development, as well as a decrease in the total leaf protein content compared to those treated with essential oils.
In our work, we have highlighted the effect of EO from Mentha piperita on durum wheat.
The results showed an increase in germination rate, average stem and root length, and total protein content in seeds treated with mint EO.
Finally, it would be interesting to continue this study to understand the behavior of durum wheat and the effect of Mentha piperita EO because they can constitute a series of future trials that can obtain a more satisfactory yield. For a better explanation and confirmation of my result, it is recommended to:
– Expand the field experiment to natural conditions to obtain more accurate results; use other varieties of durum wheat and other cereal species to determine whether the extract of Mentha piperita EO affects other varieties and species in the same way; choose different types of parameters to understand the effect of the extract better.
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